A useful research tool in neurobiology is the use of electrophysiological recording technology to monitor the electrical properties and response of a target cell in neuronal cell culture or brain tissue either in vitro or in vivo. Typically, a glass pipette is shaped to have an extremely fine point (e.g., 1-2 μm) and this is then filled with buffer and electrically connected to a recording device. This recording pipette and the target cells are visualized under a microscope using some type of direct-visible illumination. The pipette is then carefully directed to the target cell and a seal is made between the cellular plasma membrane and the glass pipette (in the case of a patch clamp recording), or the cell is pierced (in the case of whole cell recording with a sharp electrode). In the case of a patch clamp recording, gentle suction ruptures the membrane patch, preserving the seal to get undisturbed connection between the cytosol and the probe interior to isolate the cellular electrical response. Electrophysiological monitoring and recording of the cellular response can then be made over time and in response to environmental or other changes.
Given the extremely small size scale of the cells and pipette tip along with the inherently delicate nature of these biological systems, in many cases it is critical to visibly monitor the tip and targeted patch continuously during these types of experiments and assays. This allows the operator to confirm that the tip-cell seal remains intact, that the tip is correctly positioned, and that the tip and/or cells are viable and uncompromised. During in vitro recordings of brain tissue slices (typically a few 100 μm thick) or in vivo within animals, the recording pipettes/tips have to penetrate in deep tissue (over several 100 μm); here 2-photon laser illumination is typically the best, and sometimes only, technique for producing images from fluorescently labeled cells, probes and processes. Simultaneous fluorescence and visible imaging is generally impossible due to both the depth of the tissue and the nature of the recordings. Until now, the most common method used to make glass pipettes visible under such 2-photon illumination and in the tissue depth needed has been to fill the pipette with fluorescent dyes that are relatively non-toxic to these systems. These include fluorescein or AlexaFluor 594, for example. The operator then blows out or ejects dye during the approach of the tip to the cell. As living cells do not take up the dye, they can be visualized as dark “shadows” against the bright background within a given tissue. But in deeper tissue, the accumulation of such ejected dye makes the visibility poorer and complicates the recognition of cells and patching. The dyes typically used in such formats have small 2-photon action cross sections, which means that a high relative concentration of dye and a relatively high laser power typically have to be used. The dyes often are susceptible to photobleaching in these configurations and the higher laser power dramatically shortens the experimental time available as it can rapidly cause photothermal tissue damage. Additionally, there are many configurations where the addition of dye is not desired such as where cells already express some fluorescent protein-like green fluorescent protein (GFP) tags or genetically encoded calcium indicators (GECI) for Ca2+ measurements.
In view of the difficulties associated with implementation of this type of patch clamp technique, there has long been a desire to create an electrophysiological recording pipette that is itself clearly visible under 2-photon microscopic illumination. In particular, a brightly fluorescent pipette that negates the need for ejecting dye to patch labeled cells in deep tissue would be a great advantage for further exploration of in vivo cellular physiology. A need exists for a borosilicate glass pipette visible under 2-photon illumination. Techniques described herein provide the features to accomplish this with few, if any, apparent disadvantages.